This invention relates to an improved method for the coupling of antibodies to a solid phase polymeric substrate having hydroxy groups present through strong covalent chemical bonds formed by trichloro-triazine as the linking agent between the substrate and antibody. This invention also relates to the use of this substrate in immunoassay procedures for the quantitative and qualitative determination of minute quantities of various substances in body fluids such as blood, urine, saliva, etc. These immunoassay procedures may rely upon the detection and measurement of various levels of radioactivity, enzyme activity or of fluorescence in making a determination of the substances being assayed.
The use of trichloro-triazine in a procedure for covalently coupling various substances to solid substrates under anhydrous conditions for the production of solid phase catalysts and certain biospecific adsorbents is disclosed in U.S. application Ser. No. 876,240, filed Feb. 9, 1978, now U.S. Pat. No. 4,229,537.
This application has been published under International Publication No. WO 79/00609 on Aug. 23, 1979, together with an International Search Report dated May 15, 1979. These publications including the art cited are incorporated herein by reference.
The coupling or bonding of enzymes to an insoluble support sheet through an s-triazine bridge to produce an immobilized enzyme is described in U.S. Pat. No. 3,824,150. An analogous method for linking biologically active compounds to various carriers through an asymmetric bifunctional linking compound which includes an s-triazine moiety in the linking compound is disclosed in U.S. Pat. No. 4,007,089. The direct fixation of enzymes on solid support materials employing temperatures of 60.degree. C. to 120.degree. C. and an anhydrous organic liquid medium for the reaction is described in U.S. Pat. No. 4,119,494. The support materials disclosed are widely diverse and include, among others, brick, clay, sand, starch, cellulose, and various synthetic organic polymers such as polystyrene and polybutadiene, as well as copolymers of methacrylic acid. Antimicrobial compounds can be bound to a hydroxyl bearing substrate such as cellulose, starch or leather, for example, through an s-triazine bond in alkaline solution at a pH of 9 to 10 by following the procedureds described in U.S. Pat. No. 4,035,146. Cellulosic fabrics having persistent antimicrobial activity may be obtained. Various procedures have been disclosed for forming the initial s-triazine bond with the solid phase substrate or support and then coupling the activated substrate obtained with any one of several biologically active compounds. As disclosed in the patents cited above these compounds may include enzymes, antimicrobials, antigens, antibodies, haptens, etc. Depending upon the particular biologically active compound which is bound to the substrate, the resulting solid phase substrate may be employed in a wide variety of biochemical procedures including highly sensitive quantitative immunological and analytical procedures in blood chemistry. Nanogram quantities of various substances can readily be detected utilizing radio-immunoassay, enzyme immunoassay and fluorescent immunoassay techniques. The use of an activated solid phase substrate greatly simplifies these test procedures.
Of particular value for use in such immunoassay techniques are the said substrates comrising microporous membrane filters which have been activated by being coupled through an s-triazine covalent bond to a biologically active compound. Such activated materials can be produced in discrete physical forms which lend themselves quite readily to flow-through assay techniques. Here, the body fluid being tested whether blood serum, blood plasma, urine, etc. actually flows through the test system in contact with the activated surfaces of the membrane filter when carrying out the test procedure. These membrane filters are available in a range of pore sizes so that varying flow rates and contact times can be achieved within the parameters of the particular immunoassay test being used.
By coupling the activated solid phase substrate to a desired biologically active compound a wide range of immunoassay test procedures and techniques may be put to use.